1. Field of the Invention
The present invention relates to a structural gene of membrane-bound alcohol dehydrogenase complex derived from a microorganism belonging to the genus Acetobacter, and a plasmid containing the same as well as its utilization.
2. Brief Description of Related Art
A membrane-bound alcohol dehydrogenase produced by a microorganism belonging to the genus Acetobacter is an enzyme which oxidizes an alcohol into the corresponding alcohol. The enzyme takes a part in oxidative fermentation of acetic acid fermentation for producing acetic acid from ethanol, and is also utilized for quantitative determination of alcohol; the enzyme is useful from an industrial viewpoint.
Heretofore the membrane-bound alcohol dehydrogenase (hereafter simply referred to as ADH) has been obtained by culturing a microorganism belonging to the genus Acetobacter or the genus Gluconobacter, extracting and purifying from the cultured cells and has been utilized (Agricultural and Biological Chemistry, 42, 2045, 1978; ibid., 42, 2331, 1978).
For purification of this enzyme, however, fractionation by complicated column chromatography was required so that it was difficult to prepare the enzyme in large quantities. In addition, the enzyme is unstable and cannot be stored over a long period of time, which has been a problem in its application.
In order to solve these problems, it is considered to harvest mutants having an enhanced enzyme content in the cells by a mutation treatment. However, there is no report yet that any mutant having a sufficient enzyme content was harvested. It is also considered to achieve the object by cloning a gene of the enzyme and increasing the copy number of the enzyme gene or enhancing an expression degree, through genetic engineering technology. For this attempt, ADH gene of Acetobacter aceti K6033 strain has been cloned and its nucleotide sequence has been determined (Journal of Bacteriology, 171, 3115, 1989). This study is expected to be effective for improving the productivity of the enzyme by genetic engineering technology. In actuality, however, even though a plasmid carrying the enzyme gene is introduced into a host of acetic acid bacteria, the enzyme activity is not improved more than the activity inherently possessed by the host and such technique is not practical.
This is believed to be because the cloned gene would be composed only of subunits having a larger molecular weight out of the subunits constructing ADH. Any conventional ADH is purified in the form of a complex with cytochrome c from acetic acid bacteria. Matsushita et al. reported that the activity of ADH was affected depending upon the quantity of cytochrome c and cytochrome c was not present merely as an impurity but took a part in expressing the enzyme activity (Agricultural and Biological Chemistry, 53, 2895, 1989). For this reason, it was necessary to increase the subunits having a large molecular weight and at the same time, increase the amount of the subunits of cytochrome c.
Furthermore, properties of the enzyme in the cloned Acetobacter aceti K6033 strain were not studied and utility of the enzyme of K6033 strain is unclear.
In order to solve the foregoing problems, the present inventors have brought attention to ADH produced by a series of microorganisms belonging to the genus Acetobacter represented by Acetobacter altoacetigenes, which are already known to have enzymatically excellent properties, and have succeeded in cloning the structural gene of two proteins (subunits) constructing ADH and in carrying the structural gene on a plasmid.
Furthermore, the present inventors have found that by using the plasmid carrying the cloned gene, the content of this enzyme in the cells can be increased, ADH can be readily extracted and purified and, an efficiency of acetic acid fermentation can be improved. The present invention has thus been accomplished.